In age-related conditions such as Alzheimer's disease and stroke there is an increase in neuronal cell death (apoptosis and/or necrosis). In order to dissect the events of neuronal apoptosis, we have developed the first cell-free system which recapitulates the events of neuronal apoptosis, including nuclear morphological changes, internucleosomal fragmentation of DNA, caspase activation, and the proteolytic cleavage of key substrates. We have applied the system to cultured neural cells as well as to primary neurons. Using our system, we have been able to show that the bioactive peptides mastoparan and substance P, known to induce apoptosis, activate cell-free neuronal apoptosis through a process that is dependent on mitochondria. IN the research proposed here, we will use this system to examine a select set of neurodegeneratie disease-related peptides and proteins, also known to, or suspected to induce apoptosis, such as the Beta-amyloid peptides and the Prion protein, to determine whether or not their activation of apoptosis is mitochondrial dependent. Furthermore, we will use our system to determine any dependency on other organelles such as lysosomes. Finally, we will take advantage of recent work showing that Bcl-2 blocks cytochrome c release from mitochondria, and that cytochrome c and dATP, together, activate cell-free apoptosis, to extend our previous work on the roles of cytochrome c and Bcl-2 in apoptosis.